Development of small molecule drugs targeting immune checkpoints.

Immune checkpoint inhibitors (ICIs) are used to relieve and refuel anti-tumor immunity by blocking the interaction, transcription, and translation of co-inhibitory immune checkpoints or degrading co-inhibitory immune checkpoints. Thousands of small molecule drugs or biological materials, especially antibody-based ICIs, are actively being studied and antibodies are currently widely used. Limitations, such as anti-tumor efficacy, poor membrane permeability, and unneglected tolerance issues of antibody-based ICIs, remain evident but are thought to be overcome by small molecule drugs. Recent structural studies have broadened the scope of candidate immune checkpoint molecules, as well as innovative chemical inhibitors. By way of comparison, small molecule drug-based ICIs represent superior oral bioavailability and favorable pharmacokinetic features. Several ongoing clinical trials are exploring the synergetic effect of ICIs and other therapeutic strategies based on multiple ICI functions, including immune regulation, anti-angiogenesis, and cell cycle regulation. In this review we summarized the current progression of small molecule ICIs and the mechanism underlying immune checkpoint proteins, which will lay the foundation for further exploration.

Comprehensive analysis of HHV-6 and HHV-7-related gene signature in prognosis and response to temozolomide of glioma.

Human herpesvirus (HHV)-6 and HHV-7 have been detected in central nervous system and glioma tissue, while their exact role in glioma remains uncertain. Omics profiles and clinical information were downloaded from public databases, including The Cancer Genome Atlas cohort for training set and the Chinese Glioma Genome Atlas cohorts for validation sets. Differentially expressed genes between HHV-6 and HHV-7 infected or noninfected glioma patients were screened for establishing the HHV-6 and HHV-7 infection (HI) model through Lasso regression analysis. Bioinformatics methods were used to analyze the correlation between HI scores and prognosis, metastasis in glioma patients. Predictable efficacy of HI in temozolomide-resistance and HI-related genetic signatures were also explored. The HI model was constructed as: Risk score = (0.014709*DIRAS3) + (0.029787*TEX26) + (0.223492*FBXO39) + (0.074951*MYBL1) + (0.060202*HILS1). The five gene signature showed good performance in predicting survival time for glioma patients, while higher HI score is correlated with malignant features. Moreover, DNA mismatch repair genes were augmented in glioma patients with higher HI score as well as nonresponse to temozolomide treatment, which was in parallel with the transcriptomic result of temozolomide-resistant glioma cell. Targeting the five gene signature is beneficial for prognosis of glioma patients, especially in glioma patients underwent temozolomide treatment.

Macrophages in Glioblastoma Development and Therapy: A Double-Edged Sword.

Glioblastoma (GBM) is one of the leading lethal tumors, featuring aggressive malignancy and poor outcome to current standard temozolomide (TMZ) or radio-based therapy. Developing immunotherapies, especially immune checkpoint inhibitors, have improved patient outcomes in other solid tumors but remain fatigued in GBM patients. Emerging evidence has shown that GBM-associated macrophages (GAMs), comprising brain-resident microglia and bone marrow-derived macrophages, act critically in boosting tumor progression, altering drug resistance, and establishing an immunosuppressive environment. Based on its crucial role, evaluations of the safety and efficacy of GAM-targeted therapy are ongoing, with promising (pre)clinical evidence updated. In this review, we summarized updated literature related to GAM nature, the interplay between GAMs and GBM cells, and GAM-targeted therapeutic strategies.

Layered Double Hydroxides-Loaded Sorafenib Inhibit Hepatic Stellate Cells Proliferation and Activation In Vitro and Reduce Fibrosis In Vivo.

A core feature of liver fibrosis is the activation of hepatic stellate cells (HSCs), which are transformed into myofibroblasts and lead to the accumulation of extracellular matrix (ECM) proteins. In this study, we combined in vitro cellular efficacy with in vivo antifibrosis performance to evaluate the outcome of sorafenib (SRF) loaded layered double hydroxide (LDH) nanocomposite (LDH-SRF) on HSCs. The cellular uptake test has revealed that sorafenib encapsulated LDH nanoparticles were efficiently internalized by the HSC-T6 cells, synergistically inducing apoptosis of hepatic stellate cells. Moreover, the apoptosis rate and the migration inhibition rate induced by LDHs-SRF were 2.5 and 1.7 times that of SRF. Western Blot showed that the TGF-ß1/Smad/EMT and AKT signaling pathway was significantly inhibited in HSC-T6 cells treated with LDHs-SRF. For the in vivo experiment, LDHs-SRF were administered to rat models of CCl4-induced liver fibrosis. H&E, masson and sirius red staining showed that LDHs-SRF could significantly reduce inflammatory infiltrate and collagen fiber deposition and immunohistochemical results found that LDHs-SRF treatment significantly inhibited the protein expressions of α-SMA in the liver, these results suggesting that LDHs-SRF exhibited better anti-fibrotic effect than SRF alone and significantly inhibited the proliferation and activation of rat hepatic stellate cells and collagen fiber synthesis.

Fabrication and applications of bioactive chitosan-based organic-inorganic hybrid materials: A review.

Organic-inorganic hybrid materials like bone, shells, and teeth can be found in nature, which are usually composed of biomacromolecules and nanoscale inorganic ingredients. Synergy of organic-inorganic components in hybrid materials render them outstanding and versatile performance. Chitosan is commonly used organic materials in bionic hybrid materials since its bioactive properties and could be controllable tailored by various means to meet complex conditions in different applications. Among these fabrication means, hybridization was favored for its convenience and efficiency. This review discusses three kinds of chitosan-based hybrid materials: hybridized with hydroxyapatite, calcium carbonate, and clay respectively, which are the representative of phosphate, carbonate, and hydrous aluminosilicates. Here, we reported the latest developments of the preparation methods, composition, structure and applications of these bioactive hybrid materials, especially in the biomedical field. Despite the great progress was made in bioactive organic-inorganic hybrid materials based on chitosan, some challenges and specific directions are still proposed for future development in this review.

[Effects of activated CD4+ T cell-derived exosomes on cardiac remodeling after myocardial infarction].

OBJECTIVE: To explore the role of activated CD4+ T cells in cardiac remodeling after myocardial infarction (MI). METHODS: (1) Experiment in vitro: naive CD4+ T cells were isolated in mouse spleen, and then stimulated with plate-bound anti-CD3 and anti-CD28 for 48 hours. Exosomes isolated from the supernatant of activated CD4+ T cells were incubated with cardiac fibroblasts (CFs) for 48 hours, and then the ability of CFs proliferation, migration and differentiation were detected by cell counting kit-8 (CCK-8) assay, Transwell assay, and immunofluorescence assay. (2) Experiment in vivo: 40 male C57 mice were divided into 4 groups according to random number table method, including control group (Ctrl group), sham operation group (Sham group), MI group, and exosome treatment group (MI+Exo group), with 10 in each group. The mice model of MI was established by ligating the left anterior descending coronary artery. In MI+Exo group, 40 µg/d exosomes were injected intravenously into the tail after modeling. Cardiac function and cardiac fibrosis post-MI were assessed by echocardiography and quantitative polymerase chain reaction (qPCR) at 4th week. RESULTS: (1) In vitro: exosomes derived from activated CD4+ T cells significantly promote CFs proliferation, migration and differentiation [proliferation ability (A value): 0.31±0.01 vs. 0.21±0.01, migration capability (cells/MP): 79.20±3.34 vs. 48.80±2.13, differentiation ability (α-smooth muscle actin, α-SMA; fluorescence intensity): 1.56±0.03 vs. 1.00±0.02, all P < 0.05]. (2) In vivo: echocardiographic analysis showed that exosomes derived from activated CD4+ T cells aggravated the deterioration of cardiac dysfunction post-MI than MI group, as indicated by left ventricular ejection fraction (LVEF) and fractional shortening (FS) decreased significantly [LVEF: 0.185±0.008 vs. 0.257±0.022, FS: (9.72±1.72)% vs. (14.08±1.08)%, both P < 0.05], left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) increased significantly [LVEDD (mm): 5.43±0.29 vs. 4.62±0.35, LVESD (mm): 4.94±0.12 vs. 3.69±0.29, both P < 0.05]. Additionally, qPCR showed that exosomes derived from activated CD4+ T cells remarkably promoted myocardial fibrosis post-MI than MI group, as indicated by the mRNA expression of α-SMA, collagens (Col1a1, Col3a1) in MI+Exo group was significantly higher than that in MI group [α-SMA (2-ΔΔCT): 4.72±0.89 vs. 3.58±0.78, Col1a1 (2-ΔΔCT): 6.59±0.56 vs. 4.23±0.42, Col3a1 (2-ΔΔCT): 13.40±1.03 vs.4.96±0.36, all P < 0.05]. CONCLUSIONS: Activated CD4+ T cells promote cardiac remodeling following MI through transferring exosomes to CFs.

Monokine induced by gamma interferon for detecting pulmonary tuberculosis: A diagnostic meta-analysis.

BACKGROUNDS: Pulmonary tuberculosis (PTB) is an oldest-known and most formidable disease. The standard microbiology culture is time-wasting. Monokine induced by gamma interferon (MIG) has been reported as a new biomarker to auxiliarily detect PTB. In our study, we used meta-analysis to assess the diagnostic value of MIG for PTB. METHODS: PubMed, Embase, Web of Science, and Cochrane Library were searched for relative records up to April 2, 2020. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, area under the curve, and summary receiver operating characteristic curve were estimated. RESULTS: Eight studies including 1487 participants were included. The pooled sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of MIG for detecting PTB were 84%, 84%, 5.19, and 0.19, respectively. The diagnostic odds ratio and area under the curve were 27.88 and 0.90, respectively, indicating a good diagnostic ability of MIG. Meta-regression analysis showed that human immunodeficiency virus status might be a source of heterogeneity (P = .02). CONCLUSIONS: Our results showed that MIG had a good diagnostic value for PTB.

Effect of Solvent on Acyl Migration of 2-Monoacylglycerols in Enzymatic Ethanolysis.

Acyl migration occurs in many reactions and is the main obstacle for structured lipid synthesis. In this study, 2-monoacylglycerol (2-MAG) was prepared by enzymatic ethanolysis in three different media to evaluate the effect of environment on product composition. The contents of 2-MAG obtained in ethanol, hexane + ethanol, and t-butanol + ethanol systems were 30.6, 15.7, and 32.4%, respectively, after 3 h reaction. Afterward, the acyl migration kinetics of 2-MAG were studied in solvent and solventless systems without the use of lipase. Results indicate that 2-MAG in the solventless system had the highest acyl migration rate. The isomerization was efficiently prevented by the use of polar solvents, especially t-butanol. The rate constants were shown to be the highest and activation energy values were the lowest in solventless systems. The novel finding in this study was that solvent had inhibitory effect on 2-MAG isomerization, but the nonpolar hexane had the lowest inhibition of acyl migration compared to other solvents.

RANK Deficiency Ameliorates Podocyte Injury by Suppressing Calcium/Calcineurin/ NFATc1 Signaling.

BACKGROUND/AIMS: Podocyte injury and loss contribute to proteinuria, glomerulosclerosis and eventually kidney failure. Receptor activator of NF-κB (RANK) belongs to the TNF receptor superfamily, which plays a key role in the pathogenesis of podocyte injury. However, the mechanism underlying the effect of RANK in podocyte injury remains unclear. Here, we sought to explore the possible molecular mechanisms involved in podocyte injury caused by RANK. METHODS: Immortalized mouse podocytes were treated with siRNA targeting RANK for 48 h or ionomycin for 24 h before harvest. Western blot, quantitative RT-PCR and immunofluorescence staining were used to evaluate the expression and function of RANK, nuclear factor of activated T cells c1 (NFATc1), transient receptor potential cation channel, subfamily C, member 6 (TRPC6) and calcineurin in podocytes. The Calcineurin Cellular Activity Assay kit was used to detect the phosphatase activity of calcineurin in cultured podocytes. A Ca2+ influx assay was performed to analyze alterations in Ca2+ entry under different conditions. Co-immunoprecipitation assays were used to observe the relationship between RANK and TRPC6. RESULTS: RANK mRNA and protein expression were markedly increased in injured podocytes (ionomycin stimulation). Further study found that translocation of NFATc1 to the nucleus was significantly reduced after knocking down RANK by siRNA. Meanwhile, we also demonstrated that loss of RANK suppressed the phosphatase activity of calcineurin and attenuated the ionomycin-induced increase in Ca2+ influx. In addition, we showed that RANK knockdown in cultured podocytes decreased TRPC6 protein expression. Co-immunoprecipitation experiments suggested that RANK binds to TRPC6 and that ionomycin enhanced the binding of RANK to TRPC6. CONCLUSION: Our findings demonstrated that RANK deficiency ameliorates podocyte injury by suppressing calcium/calcineurin/NFATc1 signaling, which may present a promising target for therapeutic intervention.

DNA methyltransferase 1 may be a therapy target for attenuating diabetic nephropathy and podocyte injury.

The contribution of DNA methylation to diabetic nephropathy, especially the effect on podocyte integrity, is not clarified. Here we found that albuminuria in a db/db mouse model was markedly attenuated after treatment with a DNA methylation inhibitor. This was accompanied by alleviation of glomerular hypertrophy, mesangial matrix expansion, and podocyte injury. The expression of DNA methyltransferase 1 (Dnmt1), nuclear factor Sp1, and nuclear factor kappa B (NFκB)-p65 markedly increased in podocytes in vivo and in vitro under the diabetic state. The increased expression of Dnmt1 was attenuated after treatment with 5-azacytidine or 5-aza-2'-deoxycytidine or Dnmt1 knockdown, accompanied by restored decreased podocyte slit diaphragm proteins resulting from hypermethylation and improved podocyte motility. Further studies found that increased Sp1 and NFκB-p65 interacted in the nucleus of podocytes incubated with high glucose, and Sp1 bound to the Dnmt1 promoter region. The involvement of the Sp1/NFκB-p65 complex in Dnmt1 regulation was confirmed by the observation that Sp1 knockdown using mithramycin A or siRNA decreased Dnmt1 protein levels. The luciferase reporter assay further indicated that Dnmt1 was a direct target of Sp1. Thus, inhibition of DNA methylation may be a new therapeutic avenue for treating diabetic nephropathy. Hence, the Sp1/NFκB p65-Dnmt1 pathway may be exploited as a therapeutic target for protecting against podocyte injury in diabetic nephropathy.

RhoA deficiency disrupts podocyte cytoskeleton and induces podocyte apoptosis by inhibiting YAP/dendrin signal.

BACKGROUND: Podocyte apoptosis is a major mechanism that leads to proteinuria in many kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. RhoA is one of Rho GTPases that has been well studied and plays a key role in regulating cytoskeletal architecture. Previous study showed that insufficient RhoA could result in rat aortic smooth muscle cell apoptosis. However, whether RhoA is involved in podocyte apoptosis remains unknown. METHODS: Culture podocytes were treated with LPS, ADR or siRNA for 48 h before harvest. Subcellular immunoblotting, qRT-PCR, immunofluorescence and flow cytometry were used to exam the expression and function of RhoA or YAP in podocytes. RESULTS: We found that the expression of RhoA and its activity were significantly decreased in LPS or ADR-injured podocytes, accompanying loss of stress fibers and increased cell apoptosis. Knocking down RhoA or its downstream effector mDia expression by siRNA also caused loss of stress fibers and podocyte apoptosis. Moreover, our results further demonstrated that RhoA deficiency could reduce the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Silenced dendrin expression significantly abolished RhoA, mDia or YAP deficiency-induced podocyte apoptosis. CONCLUSION: RhoA deficiency could disrupt podocyte cytoskeleton and induce podocyte apoptosis by inhibiting YAP/dendrin signal. RhoA/mDia/YAP/dendrin signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary RhoA would be one potent way to prevent proteinuria kidney diseases.

Spironolactone inhibits podocyte motility via decreasing integrin ß1 and increasing integrin ß3 in podocytes under high-glucose conditions.

Integrin ß1 and ß3 expression by podocytes is required to maintain glomerular structural integrity. Previous studies have shown that aldosterone (ALD) is involved in glomerular podocyte injury, and mineralocorticoid receptor (MR) blocker spironolactone effectively reduces proteinuria in patients with diabetic nephropathy. The present study was designed to observe the effects of spironolactone on ß1 and ß3 integrin expression and podocyte motility under in vitro diabetic conditions. Immortalized mouse podocytes were cultured in media containing normal glucose (NG) levels, high glucose (HG) or HG plus spironolacton. The expression of ß1 and ß3 integrin in podocytes was detected by reverse transcription quantitative polymerase chain reaction, immunofluorescence and western blot analyses. The effects of spironolacton on podocyte motility was further evaluated using a wound healing assay. HG stimulation markedly decreased mRNA and protein expression of integrin ß1, and significantly increased mRNA and protein expression of integrin ß3 in cultured podocytes. However, simultaneous treatment with spironolacton (10­7 mol/l) significantly attenuated HG-mediated increases in integrin ß3 and decreases in integrin ß1 expression. Furthermore, the migration of podocytes induced by HG was abrogated by concomitant treatment with spironolacton. In conclusion, the present study suggested that HG decreased the expression of integrin ß1 in cultured podocytes, accompanied with an increase of integrin ß3. Spironolactone inhibited cell motility and stabilized podoctyes treated with HG, probably through partly normalizing the expression of integrin ß1 and decreasing the expression of integrin ß3.

[Evaluation of preparation of curved root canals using hand-used ProTaper].

OBJECTIVE: To evaluate the shaping ability of hand-used ProTaper on curved canals using Endodontic Cube. METHODS: Fifty-four curved root canals in vitro were selected and divided into three groups according to the curved degree (alpha), group A: 0 degrees < or = alpha < 25 degrees , group B: 25 degrees < or = alpha < 40 degrees , group C: 40 degrees < or = alpha < 55 degrees . Endodontic Cube was assembled, and each sample was sectioned perpendicular to the axis of the tooth into four sections with Isomer-Buhler in low speed. Then the root canals were prepared with hand-used ProTaper. Before and after shaping, photograph of all the sections were taken under a stereomicroscope. Statistical analyses were performed. RESULTS: The dentin cutting quantity of the whole canal prepared with ProTaper in group B and C was larger than that of group A. The deviation distance of the whole canal prepared by ProTaper in group C was significantly larger than that in group A, and the deviation distance in middle portion larger than that in group B. The maintaining ability in the middle portion of group C by ProTaper was worse than that of group A and B. CONCLUSIONS: The curvature of root canal may increase the cutting quantity of the -dentin and reduce the ability of remaining original canal shape prepared by ProTaper.